1. Field of the Invention
The present invention relates to a method for searching for a variety of new physiologically active substances and producing the same.
2. Background Art
Physiologically active substances of unknown types have been searched, heretofore, by analyzing substances present in body fluids or tissues, and identifying and separating new substances therefrom to determine the physiological activities of said substances.
The prior art method as described above comprises the steps of analyzing components in living organisms, searching and isolating new substances, and identifying the physiological activities thereof. It is obvious, however, that there exist an extremely large number of substances within a living organism, and many physiologically active substances are often present at very low concentrations, thus making it difficult to isolate new substances. Furthermore, because living organisms perform a number of physiological activities, it may also be difficult to identify the physiological activities of a newly isolate substance. As can be appreciated from the above discussion, the prior art methods render it difficult to search and isolate new physiologically active substances.
One aspect of the present invention is to provide a method for searching, with more efficiency and a certain degree of predictability, new physiologically active substances.
The inventor of the present invention found that in the case of a receptor satisfying the following condition below, either X or Y, and also having one or more variants in size because of alternative splicing or post-translational modification, the amino acid sequence of the missing portion, i.e., the spliced portion or the portion inserted or added, has a physiological meaning. Condition X is the condition where there is in the body a substance or cell having a functional antagonism against a substance A which is a ligand of a receptor. Condition Y is the condition where there is in the body a substance or cell having a functional antagonism against a cell which expresses the receptor whose ligand being a substance A. For example, calcitonin may be bound to a calcitonin receptor present on osteoclasts to suppress the deossification effected by the osteoclasts, while there are osteoblasts that have functional antagonism against the osteoclasts. Regarding calcitonin receptors, there are already reported the amino acid sequence thereof, but there are reported two or more different types having different sizes. The inventor of the present invention has predicted that, in case of calcitonin receptors of such types having two or more sizes, one domain in the receptor of longer size is spliced thereby creating the receptor of shorter size, and that said spliced domain may cause effects on osteoblasts having effects antagonistic to the effects of osteoclasts on which calcitonin causes effects. Upon examination of the spliced domain after chemically synthesizing the domain, it was confirmed that this new peptide promotes osteogenesis by binding to receptors on the osteoblasts. Such confirmation as above showed the correctness of the foregoing prediction made by the inventor of the present invention and the present invention was made.
In another words, the present invention provides a method for searching physiological active substances, the method comprising the steps of identifying an amino acid sequence of receptors which have two or more sizes when the receptors are functionally identical to each other, the receptor being a receptor of a substance when there is present in human body a substance or cell which have a functional antagonism, or the receptor being a receptor of substance when there is present in human body a cell or substance which have functional antagonism to cells on which said substance causes some effects, and determining which domain in the receptor of longer size is missing in the receptor of shorter size. The present invention also provides a method for producing physiologically active peptides, the method comprising the step of producing the missing domain, or the derivative thereof, identified by the method of present invention as described above.
One aspect of the present invention provides for the first time a method for searching, with efficiency and a certain degree of predictability, for new physiologically active substances. According to another aspect of the present invention, new physiologically active substances may be identified by analyzing receptors of substances associated with functional antagonism, thereby eliminating the necessity of isolating, as before in the prior arts, physiologically active substances present in a small quantity in a sample of human body which includes an extremely large number of components. Furthermore, because the physiological activity of the identified physiologically active substance is associated with said functional antagonism, it may be much easier than before to identify the physiological activities thereof. Therefore, according to one method of the present invention, it is made possible to identify new physiologically active substances in much more efficient way than before.
According to one method of the present invention, focus is made on receptors, the receptor being a receptor of a substance when there is present in human body a substance or cell which have a functional antagonism, or the receptor being a receptor of substance A when there is present in human body a cell or substance which have a functional antagonism to cells on which the substance A causes some effects. The functional antagonism is a fundamental function for the homeostasis in human body and there are a large number of substances or cells having mutual antagonisms within human body. Examples of receptors, the receptor being a receptor of a substance when there is present in human body a substance or cell which have a functional antagonism, or the receptor being a receptor of substance A when there is present in human body a cell or substance which have a functional antagonism to cells on which the substance A causes some effects, may be found in many receptors, such as Calcitonin receptors (osteoclasts on which calcitonin acts shows functional antagonism to osteoblasts), glucagon receptors (glucogan having functional antagonism to insulin), somatostatin receptors (somotostatin having functional antagonism to growth hormones), and parathyroid hormone (the hormone having functional antagonism to calcitonin, etc.) and the like. Such receptors include but are not limited to 7-transmembrane type receptors such as Carcitonin receptors.
According to one method of the present invention, the amino acid sequences or the sizes of such receptors are analyzed to find receptors having different sizes and yet being identical receptors. This process may be performed by identifying a plurality of times the amino acid sequences or the sizes of the identified receptors, or by making use of literature if reported therein. Some examples of receptors, wherein there are receptors having two or more types having different sizes while being identical receptors may be found in calcitonin receptors, glucagon receptors, somatostain receptors, and the like.
After identifying that there are two or more types of receptors having different sizes for identical receptors, the amino acid sequences of these two or more types of receptors are compared with each other to identify which domain in the receptor of longer size is missing and thereby rendering it the receptor of shorter size. The missing domain in the receptor of shorter size is the physiologically active substance having some effects on the functional antagonism. In another words, some of the elements in the spliced structure are the physiologically active substances. It may be concluded that a physiologically active substance is bound to a receptor whereby some portion of the receptor is spliced and this spliced piece of the receptor shows some physiological activity, such as a controlling effect on the functional antagonism. It was confirmed, for example, that, as shown in the examples below, the missing domain in a calitonin receptor is bound to a receptor present on a osteoblast thereby promoting osteogenesis.
Since the missing domain identification by the above-described method has physiological activities, a physiologically active substance may be obtained by producing such domain. In most cases, the missing domain comprises peptides of relatively short sizes, and therefore, in such cases, a commercially available peptide synthesizer may be used to easily produce chemically synthesizing the physiologically active substances. Alternatively, such substances may be produced by using a method of gene engineering type according to known methods.
The physiological activity of the physiologically active substance thus obtained is related to the above-mentioned functional antagonism, and therefore, the activity can be easily confirmed by any of suitable methods applicable depending on each functional antagonism.
It is well-known to those skilled in the Art that, in peptides having physiological activities in general, the physiological activity thereof may be maintained even when some of the amino acids thereof are substituted by other amino acids, or when some amino acids are added, or when some of the amino acids are missing. Therefore, the present invention includes a method for producing a substance which has physiological activities inherent to physiologically active substances comprising the said missing domain, wherein some of the amino acids constituting the missing domain are replaced with other amino acids, or some other amino acids are added to amino acids constituting the missing domain, or some of the amino acids are missing from the amino acids constituting the missing domain. (Foregoing substance is referred to hereinafter in the present application as xe2x80x9cderivativexe2x80x9d of the missing domain.) Such a derivative preferably has not less than 70%, and more preferably 90%, of homogeneity with the above-mentioned missing domain.